The present invention is directed to molecular imprints able to selectively bind macromolecules. The molecular imprints of the present invention can be prepared without obtaining a purified sample of a target macromolecule. A template molecule possessing the structure of a portion of the macromolecule is synthesized and then imprinted. Molecular imprints made by this method form selective complexes with their target macromolecules. Arrays of molecular imprints can be used to rapidly and inexpensively screen diverse biological samples.
The recent explosion in the number of novel macromolecules, many identified by the genome sequencing efforts, has intensified the need for improved compositions and methods for separating macromolecules. Thousands of recently identified macromolecules have yet to be purified and characterized functionally. Techniques for the rapid capture, isolation, detection, analysis, and quantification of macromolecules would accelerate the functional characterization of novel macromolecules.
In particular, when the macromolecule is a protein, current methods of capture and separation are cumbersome and expensive. In one current technique, affinity matrices are used to capture and/or separate a protein of interest from a mixture of proteins and other molecules. Affinity matrices might be prepared using a purified sample of the protein to create antibodies. However, the preparation of antibodies that specifically bind a protein can take several months and might even require a purified sample of the protein. Alternatively, an affinity matrix might be prepared using knowledge of the function of the macromolecule. For instance, an affinity matrix based on a binding partner of the protein might be used for capture and separation of the protein. Unfortunately, methods of separating macromolecules that require extensive knowledge about the macromolecule, or even a purified sample of the macromolecule, are ineffective with macromolecules that have yet to be characterized. The requirement of a purified sample of the macromolecule for the preparation or selection of an affinity matrix often presents researchers with nothing more than frustrating circularity.
In addition to affinity matrices, other techniques are also used to separate macromolecules. For example, the current state of the art technique for separating large numbers of proteins is two-dimensional gel electrophoresis. The technique typically resolves about 2,000 proteins, and the best gels can resolve up to 11,000 proteins (Abbot, 1999, Nature 402:715-720). Unfortunately, many researchers require separation techniques that can resolve proteins from samples as diverse as the entire protein fraction of a mammalian cell. The protein fraction of a cell can contain tens of thousands of proteins, overwhelming the resolving power of 2D electrophoresis (Abbot, 1999, supra). Since the full sequences of the genomes of many species, including humans, are nearing completion, researchers must now grapple with the functions of hundreds of thousands of novel macromolecules (Abbot, 1999, supra). New techniques of macromolecular separation that require limited information or even no information about the target macromolecules are needed.
Currently, researchers are seeking improvements in protein separation. For instance, some are attempting to create chips which specifically bind proteins. In one typical chip, antibodies specific for known proteins are attached to a substrate to form a microarray. These chips can then be used to bind and identify proteins from a complex solution (Abbot, 1999, supra). However, these chips suffer from the same limitations of antibody production that plague affinity matrices. For each protein to be bound by the chip, a unique antibody must be prepared by an expensive process that can take several months. In addition, many proteins are not sufficiently immunogenic to create antibodies for binding.
In the field of small molecules, the technique of molecular imprinting has provided an efficient method for the preparation of matrices that are capable of selectively binding a target molecule. To prepare a molecular imprint, a matrix is formed around a template molecule. After the matrix has formed and the template molecule is removed, the matrix can then be used to selectively bind the template molecule. As early as 1949, a silica gel was created that selectively bound a dye (Dickey, 1949, Proc. Natl. Acad. Sci. USA 35:227-229). Recently, an imprint prepared with phenyl-xcex1-D-mannopyranoside was sufficiently selective to resolve a racemic mixture of the saccharide (Wulff, 1998, supra).
Current methods form imprints of molecules in organic polymers (Wulff, 1998, Chemtech 28:19-26). To create cavities of defined shape, polymerizable molecules are bound, covalently or noncovalently, to a template molecule (Wulff, 1998, supra). The resulting complex is copolymerized in the presence of a large amount of a cross-linking reagent (Wulff, 1998, supra). The templates are then removed leaving microcavities with defined shapes and arrangements of functional groups (Wulff, 1998, supra). Imprints made by such a technique display selective binding for the template molecule. Molecular imprints have been used for chromatographic separation, immunoassays, chemosensors, and even catalysis (Wulff, 1998, supra).
To date, molecular imprints have had limited application to the binding of larger molecules including macromolecules. In fact, one review states that only small molecules can be imprinted with any great confidence. Molecular imprints of larger molecules like nucleic acids, peptides, proteins and cells fail because larger molecules yield more heterogeneous binding sites and because larger molecules can be too fragile for conventional methods of molecular imprinting (Cormack and Mosbach, 1999, Reactive and Functional Polymers 41:115-124).
Nevertheless, a few successful imprints of larger molecules have been produced. Synthetic polymers which selectively bind amino acid derivatives and peptides were created using the target amino acid derivative or peptide as a template (Kemp, 1996, Anal. Chem. 68:1948-1953). Imprints have also been created which bind to nucleotide derivatives (Spivak and Shea, 1998, Macromolecules 31:2160-2165). Ionic molecular images of polypeptides have been created by mixing a matrix material with the intact polypeptide chain to be bound by the molecular image (U.S. Pat. No. 5,756,717). Molecular imprints of cytochrome c, hemoglobin and myoglobin, respectively, have been prepared by polymerizing acrylamide in the presence of each intact protein (U.S. Pat. No. 5,814,223). An imprint of horse myoglobin selectively bound horse myoglobin from a mixture of proteins including whale myoglobin (U.S. Pat. No. 5,814,223).
Although current methods of molecular imprinting have shown limited initial success at selectively binding macromolecules, the current methods are not sufficient for the efficient capture of macromolecules. The current techniques for molecular imprinting require a purified sample of the macromolecule to be bound by the imprint. The inability to produce a specific imprint in the absence of a purified sample of the macromolecule is no different from one of the failings of conventional methods of protein separation. In addition, current methods of preparing molecular imprinting are not amenable to creating the thousands of imprints often required by current large-scale experiments. Purification of hundreds or thousands of proteins to create a matrix for separating the proteins of a cell extract is no more efficient that 2D electrophoresis. An efficient method for producing compositions that selectively bind macromolecules given limited information about the structure or function of those macromolecules is needed. An ideal method could produce a composition capable of binding a macromolecule given as little information as a partial primary structure of the macromolecule.
An improvement in molecular imprinting to enable the preparation of affinity matrices in the absence of a purified sample of the macromolecule would overcome many limitations of the art of molecular imprinting. Techniques are also needed to efficiently separate and identify thousands of proteins. Compositions with specificity for macromolecules that can be produced rapidly and at low cost will enable such techniques. Ideal compositions would be arrays of such binding compositions, each composition designed to bind a given macromolecule. Such an array could be used to rapidly screen a complex biological sample for a number of different macromolecules simultaneously.
These and other shortcomings in the art are overcome by the instant invention, which in one aspect provides imprint compositions useful for capturing, isolating, detecting and/or quantifying macromolecules in a sample. Generally, the imprint compositions comprise a matrix material defining an imprint of a template molecule. The template molecule typically corresponds to a portion of a macromolecule of interest, such as, for example, a polynucleotide, a polypeptide, polysaccharide, a protein, a glycoprotein, a receptor, an enzyme, a nucleic acid, a carbohydrate, etc. If the macromolecule is composed of n identifiable structural units as defined below, then the template molecule can correspond to a portion of the macromolecule that includes from 1 up to (nxe2x88x921) of those structural units. Alternatively, the template molecule can correspond to as little as 1% of the macromolecule or to as much as 99% of the macromolecule. The portion to which the template molecule corresponds may be an internal portion of the macromolecule or a terminal portion of the macromolecule. Alternatively, the portion may be a side-group or modification of the macromolecule, such as a polysaccharide group of a glycoprotein macromolecule, or a portion thereof. Preferably, the template molecule will correspond to a contiguous terminal portion of the macromolecule.
Matrix materials that can comprise the imprint compositions of the invention include substances that are capable of undergoing a physical change from a fluid state to a semi-solid or solid state. In the fluid state, the particles of a matrix material move easily among themselves, and the material retains little or no definite form. A matrix material in the fluid state can be mixed with other compounds, including template molecules. In the semi-solid or solid state, the matrix materials are capable of forming and retaining cavities that complement the shape of template molecules. Examples of such matrix materials include heat sensitive hydrogels such as agarose, polymers such as acrylamide, and cross-linked polymers.
The imprint compositions of the invention may take a variety of different forms. For example, they may be in the form of individual beads, disks, ellipses, or other regular or irregular shapes (collectively referred to as xe2x80x9cbeadsxe2x80x9d), or in the form of sheets. Each bead or sheet may comprise imprint cavities of a single template molecule, or they may comprise imprint cavities of two or more different template molecules. In one embodiment, the imprint composition comprises imprint cavities of a plurality of different template molecules arranged in an array or other pattern such that the relative positions of the imprint cavities within the array or pattern correlate with their identities, i.e. the identities of the template molecules used to create them. Each position or address within the array may comprise an imprint cavity of a single template molecule, or imprint cavities of a plurality of different template molecules, depending upon the application. Moreover, the entire array or pattern may comprise unique imprint cavities, or may include redundancies, depending upon the application.
As discussed above, the template molecule used to make the imprint will typically correspond to a portion of a macromolecule of interest. However, as will be discussed more thoroughly below, an important aspect of the invention includes the ability to use the imprint compositions of the invention to isolate novel macromolecules from complex mixtures and/or samples. In this embodiment, a template molecule can have a structure that does not necessarily correspond to a portion of any known macromolecule. Rather, the template molecule could have a structure that corresponds to a portion of a consensus sequence derived from a family of macromolecules. Alternatively, the template molecule might have a random structure. A molecular imprint of a template molecule can bind a novel macromolecule if the template molecule corresponds to a portion of the novel macromolecule. An array of imprints of template molecules can be used to rapidly screen a mixture for novel macromolecules. An array of imprints of the complete set of polymeric template molecules composed of a defined number of monomers can be used to capture most or all of the macromolecules of a mixture.
In another aspect, the present invention provides methods of making the imprint compositions of the invention. According to the method, a compound or mixture of compounds that is capable of undergoing a change of physical state such that the resultant product is a solid or semi-solid matrix capable of retaining shaped cavities is contacted with a template molecule under conditions in which the change of physical state is effected. Changing the physical state of the compound or mixture of compounds in the presence of the template molecule results in a solid or semisolid matrix having the template molecules entrapped therein. The template molecules are then removed, yielding a solid or semisolid matrix defining cavities that correspond in shape to the template molecules. This resultant product is a molecular imprint composition. Particularly preferred methods of making molecular imprint compositions include the method of surface imprinting described in copending application Ser. No. 09/507,299, filed concurrently herewith, which is incorporated herein by reference.
In still another aspect, the present invention provides methods of using the imprint compositions to capture, isolate, detect, analyze and/or quantify a macromolecule of interest in a sample. According to the method, a sample suspected of containing a macromolecule of interest is contacted with an imprint composition of the invention under conditions in which the macromolecule binds the imprint composition. The imprint-macromolecule complex may be optionally rinsed to remove unbound components of the sample. The macromolecule may be dissociated from the complex and isolated and/or quantified. Alternatively, the presence of the macromolecule may be detected, and/or its quantity determined, without dissociating it from the complex.
The methods can be used to capture macromolecules of known, partially known or unknown structure. In the former two embodiments, the imprint composition comprises an imprint of a template molecule that corresponds to a known portion of the macromolecule of interest. In the latter embodiment, the imprint may comprise an imprint of a template molecule that corresponds to a conserved portion of a specific class of macromolecules, such as for example, a conserved portion of a receptor superfamily or family, or it may comprise a predicted sequence or a completely random sequence. These latter imprint compositions can be used to capture and/or isolate novel members of known classes of macromolecules, or completely new types of macromolecules.
Macromolecules may be detected, captured, isolated, analyzed and/or quantified according to the methods of the invention singly, using an imprint composition specific for a particular macromolecule of interest, or alternatively, pluralities of different macromolecules can be captured simultaneously from a complex mixture using, for example, the array or pattern imprint compositions described herein, for subsequent detection, isolation and/or quantification.
The methods and compositions of the invention provide significant advantages over currently available protein separations and molecular imprinting technologies. Unlike known imprinting techniques, the molecular imprints of the present invention do not require a purified sample of a target macromolecule for preparation. The primary structure of a portion of the macromolecule is sufficient to create an imprint that can specifically capture the macromolecule. If the target macromolecule is novel, a molecular imprint of template molecules that do not necessarily correspond to portions of known macromolecules can be used to screen for the target macromolecule. Because they do not require isolation of the macromolecule of interest, the molecular imprints of the present invention can be prepared in far less time and at a fraction of the cost of conventional protein separation media.
The methods and compositions of the invention also have widespread applicability, ranging from the detection and/or isolation of specific macromolecules of interest from samples, to the capture, isolation, analysis and/or quantification of pluralities of macromolecules from complex mixtures for applications such as, for example, expression profiling, to the discovery of novel members of known classes of macromolecules and/or completely new types of molecules altogether.